Plants are often simultaneously exposed to soil drying (drought) and high-temperature stress conditions. Drought is one of the most widespread environmental variables affecting growth and development of plants. Among the prominent effects of drought stress on plant physiology and metabolism are reductions of photosynthesis, photosynthate translocation, transpiration, protein synthesis, and cell wall synthesis. Changes in gene expression also occur in response to drought stress. In addition, drought stress modifies cellular ultrastructure, including injuries to membranes.
Temperature also plays an important role in the physiological processes of plants. Increased temperatures that rise to the level of heat shock or heat stress affect cell metabolism, causing changes in the rates of biochemical reactions. Elevated temperatures further reduce photosystem II activity, photophosphorylation, photosynthetic enzyme activity, dark respiration, protein synthesis, and ion uptake. Increased temperatures also cause injuries to cellular membranes. The molecular bases of such injuries are denaturation and aggregation of proteins and formation of hexagonal II, a non-bilayer lipid phase.
The reduction in photosynthetic activity in plants associated with heat and drought stress is primarily attributable to chloroplast damage. Leaf dehydration and high temperatures can severely disrupt the ultrastructure of chloroplasts. The main damage to the chloroplast caused by water stress includes structural changes resulting from excessive swelling, distortion of the intergranal and granal lamellae, and the appearance of lipid droplets. [Poljakoff-Mayber, A (1981) Ultrastructural consequences of drought, pp. 389-403 in L. G. Paleg, ed. The physiology and biochemistry of drought resistance. Bot. Gaz. 152:186-194.] Damage to the chloroplast caused by high temperature mostly comes from detrimental effects on chloroplast envelope membranes and thylakoid membranes.
Drought and high temperatures are major limiting factors to plant productivity, often causing significant economic losses to U.S. agriculture. According to the American Association of Nurserymen, 30% of all one- and two-year-old field grown plants were lost in the Midwestern states. Cosgrove T (1988b) The industry's year in review. American Nurseryman 169:31-37. Numerous growers who were without irrigation lost 50% or more of their crops. Even those with on-site irrigation were unable to counter the relentless, record-breaking heat.
Indirect costs of high-temperature stresses are also noted in the costs associated with the installation and use of irrigation equipment on high value crops. Virtually all climatologists agree that high-temperature stresses will intensify due to the “greenhouse effect”. Cosgrove T (1988c) Summer droughts and the “greenhouse effect”. American Nurseryman 168:23-33. Consequently there is an increasing need in the art for new cultivars that have increased tolerance to heat stress and drought conditions to improve crop yields.
Traditional methods of improving plant heat tolerance have centered around breeding techniques. While improvements have been achieved, breeding techniques are laborious and slow. Further breeding strategies have been hampered since plant heat tolerance is a complex characteristic that is difficult to evaluate, which limits selection procedures. Thus, it would be desirable to utilize recombinant DNA technology to produce new plant varieties and cultivars in a controlled and predictable manner. To increase yield it would be especially desirable to produce crop and ornamental plants with improved tolerance to stress over a range of environmental conditions.
It can be seen from the foregoing that a need exists in the art for a transgenic method of increasing yield potential in crop and ornamental plants by improving tolerance to stresses caused by heat and drought conditions.
A rise in temperature above a certain level may result in the death of the plant. Levitt recognized the so-called heat-killing temperature as the temperature at which 50% of the plant is killed. Levitt J. (1980) Responses of Plants to Environmental Stress. Water, radiation, salt, and other stresses, 2. Academic Press, New York. However, plants exposed to sublethal high temperatures have been shown to acquire thermotolerance to otherwise lethal high temperatures. Chen H H, et al. (1982) Crop Sci 22:43-47. Specifically, a temperature shift of 8-10° C. above the normal growing temperature induces the synthesis of a set of new proteins, known as heat-shock proteins (HSPs). Lindquist S (1986) Ann Rev Biochem 55:1151-1191. The synthesis of HSPs has been observed in a variety of plant species, and the general phenotype of the heat shock response is highly conserved in all organisms. Id.
The conservative nature of HSPs and their synthesis under elevated temperatures suggest their involvement in heat resistance. Correlations between heat resistance acquired from heat pretreatments and synthesis of HSPs have been found in many species. Altschuler M., et al. (1982), Plant Mol Biol 1:103-115. In addition, recent studies have shown that specific HSPs are absolutely required for the establishment of heat resistance. Lee Y R J, et al. (1994), Plant Cell 6:1889-1897. It is generally thought that HSPs play an important role in the development of heat resistance by acting as molecular chaperones. Ellis J. (1987), Nature 328:378-379. Molecular chaperones are involved in the stabilization of proteins in a particular state of folding.
Several studies have revealed qualitative differences in the synthesis of HSPs between genotypes that differ in drought and/or heat tolerance. The heat-tolerant Triticum aestivum L. cv. Mustang synthesized unique HSPs that were absent in the heat-sensitive T. aestivum cv. Sturdy (Krishnan et al., 1989). Qualitative differences in the synthesis of HSPs have also been observed between the heat-tolerant Gossypium barbadense and heat-sensitive G. hirsutum (Fender and O'Connell, 1989). Differences in the profile of HSPs were also found between drought tolerant Lycopersicon pennellii and drought susceptible L. esculentum (Fender and O'Connell, 1990).
A recent study has revealed a genetic relationship between heat tolerance and the synthesis of specific HSPs (Park et al., 1996). A heat tolerant variant of Agrostis palustric Huds. synthesized heat shock polypeptides of 25 kb (HSP25) which were absent in a heat sensitive variant. Analysis of the F1 progeny from these variants revealed a positive correlation between the ability to synthesize HSP25 and thermotolerance.
Few other genetic studies have been undertaken to investigate possible associations of HSPs with drought and/or heat tolerance. Further, the studies that have been conducted have not demonstrated an association between the HSPs tested and drought and/or heat tolerance. For example, when the heat-tolerant Gossypium barbadense was crossed to heat-sensitive G. hirsutum, the unique HSPs of G. barbadense did not associate with the heat-tolerant phenotype (Fender and O'Connell, 1989). Similarly, an interspecific cross between drought tolerant Lycopersicon pennellii and drought susceptible L. esculentum showed no association of HSPs with drought tolerance (Fender and O'Connell, 1990).
The failure of previous experiments to demonstrate association of HSPs with drought and/or heat tolerance is not surprising. Drought and heat tolerance are complex characteristics, and many factors can affect the plant's ability to tolerate stress (Levitt, 1980a, 1980b). Inability of a plant to synthesize one or few specific HSPs might be compensated by other factors that are involved in the tolerance to drought and/or heat stress.
Protein synthesis elongation factor (EF-Tu) has been intensely studied for many years in relation to its role in which peptides are elongated on ribosomes. EF-Tu is a protein of 45 kD which is involved in the elongation of polypeptides during the translational process of protein synthesis. Riis et al. (1990), Eukaryotic protein elongation factors, TIBS 15:420-424. EF-Tu is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. EF-Tu is one of the most abundant proteins in rapidly growing Escherichia coli cells, with approximately 5-6 copies per ribosome. Kudlicki, W. (1997), Renaturation of Rhondanese by Translational Elongation Factor (EF) Tu, J Biol Chem 272:32206-32210.
Bacterial EF-Tu has been reported to interact with unfolded and denatured proteins in a manner similar to molecular chaperones that are involved in protein folding and protein renaturation after stress. Caldas, T. (1998), Chaperone Properties of Bacterial Elongation Factor EF-Tu, J Biol Chem 273:11478-11482. The major classes of bacterial chaperones comprise DnaK/Hsp70 (and its assistants DnaJ and GrpE), GroEL/Hsp60 (and its assistant GroES), HtpG/Hsp90, and the heat shock proteins.
The present inventors purified and isolated a novel maize EF-Tu protein and have surprisingly discovered an association between the synthesis of increased levels of EF-Tu and increased tolerance to drought and heat in maize. This chloroplast EF-Tu has been found to play a role in the development of drought and heat resistance in maize by increasing heat stability of chloroplasts. This discovery may be used in the creation of new varieties of crop plants which display increased tolerance to heat stress.
It is therefore an object of the present invention to provide a novel isolated, purified and characterized EF-Tu protein from maize. It is a further object to provide expression constructs which provide for temporal and spatial expression of EF-Tu in a transgenic plant, to increase resistance to stress through heat stability of chloroplasts.
It is yet another object of this invention to provide transgenic plant lines with heritable phenotypes which are useful in breeding programs designed to increase heat and drought tolerance in crop plants over a range of environmental conditions.
It is yet another object of this invention to produce seed which will produce plants with increased yield tolerance to heat and drought stress.
It is yet another object of this invention to provide plants, plant cells, and plant tissues containing the expression constructs of the invention.
Other objects of the invention will become apparent from the description of the invention which follows.